The 13 KDa seed-specific promoter together with relative 5’ UTR and transit peptide sequence (NCBI acc. N° D63901) was amplified from Oryza sativa var. Ariete using a forward and reverse primer (PROL-fw and PROL-rv, table 1). The 729 bp promoter fragment was inserted in pGEM-T (Promega, Madison WI USA), and the accuracy of the amplified promoter region was verified by DNA sequencing30. A PstI and XbaI site was inserted respectively in the forward and reverse primer to ease the subsequent DNA cloning in a plant expression vector (pCAMBIA1302, NCBI Acc. N° AF234298) in PstI and XbaI. The cDNA of mature ApoA-IM obtained according to Romano et al.(2018)19 was put under the control of the rice 13 KDa prolamin promoter and cloned respectively in BamHI and SacI (blunted) (Figure 1a).
Rice transformation and analysis of transgenic plants.
Embryonic calli derived from mature zygotic embryos of the rice (Oryza sativa var. Rosa Marchetti) were inoculated with Agrobacteriumtumefaciens strain EHA105 transformed by electroporation with the plasmid pCAMBIA-PROL-ApoA-IM. Callus and bacterial induction, transformation, selection, and regeneration of transgenic tissues were performed as described in Hiei et al. (1994)31, with minor modifications. Putatively transformed (hygromycin-resistant) plants (T0 plants) were potted in peat and hardened in a greenhouse together with controls (WT rice) to produce T1 seeds. These T1 seeds were then subsequently planted to produce T1 plants and T2 seeds. During the entire experimental period, the plants were evaluated for the principal agronomic characteristic.
PCR Analysis. Before flowering, T0 plants were subjected to PCR analysis to verify the presence of the transgene. The PCR was done using DNA extracted32 from leaves of the rice lines resistant to hygromycin, using the primers Apo-fw and Apo-rv (Table 1), which amplify the entire Apo A-I gene, visualized, in the case of successful transformation, by a fragment of 732 bp.
Southern Analysis. Independent transgenic rice plants expressing the ApoA-IM gene were subjected to Southern Blot analysis to verify the copy number and insertions of the transgene present in each plant. The total genomic DNA was extracted from young leaves according to Doyle and Doyle (1987)33. Approximately 10 µg of DNA was digested with XbaI which cuts only once in pCAMBIA-PROL-ApoA-IM, fractionated on a 0,8% (W/v) agarose gel, transferred to a positively charged nylon membrane, and hybridized with a probe corresponding to ApoA-I M CDS labelled with the PCR DIG Probe Synthesis Kit (Roche Molecular Biochemicals Penzberg Germany) following the manufacture’s instruction. The primer set used in PCR analysis was also used to generate the DIG-labelled probe. The positive control consisted of the vector (pGEMT-APOA-IM) linearized with the same restriction enzyme. The detection was realized with CDP-Star® (GE Healthcare, Chicago Illinois USA) following the manufacturers’ instructions.
Evaluation of protein expression
The seeds collected from the T0 and T1 plants PCR positive were then used to verify the expression by Western Analysis. Briefly, the total protein from rice seeds (100 mg) was extracted from the seeds using extraction buffer (50 mM Tris-HCl pH 8.0, 5 mM EDTA, 200 mM NaCl, 0.1% Triton X-100, 1mM PMSF) in ratio 1:10 (w/v), mixed with SDS-loading buffer (with or without β-mercaptoethanol, to discriminate from monomer/dimer respectively), subjected to electrophoretic separation on an acrylamide gel, and transferred using electroblotting (solution of 25 mM Tris, 192 mM glycine, 20% methanol, 30 V at 4°C overnight) to a nitrocellulose membrane, hybond ECL (GE Healthcare, Chigaco Illinois USA). The membrane with the bound protein was placed in a solution of PBS-T and 5% skim milk, agitated for 60 minutes, washed, then exposed to the primary antibody Anti ApoA-I Goat 1:5000 (Acris, San Diego CA, USA) and subsequently to the secondary antibody Anti-Goat peroxidase conjugate, 1:12.000 (Sigma Aldrich, St. Louis Missouri USA). The membrane was washed several times and placed in a chemiluminescent detection solution, ECL (GE Healthcare, Chicago Illinois USA).
Protein quantification in rice seeds. An indirect competitive ELISA (IC-ELISA) was used to detect ApoA-IM protein in rice seeds. In particular, recombinant hApoA-I (Sigma Aldrich St. Louis Missouri USA) was coated (600ng/ml) onto a micro-well plate overnight at 4°C, after which the plate was washed three times with 0,01 M PBS (pH 7) and then blocked with 200 μl of 5%(W/V) BSA for 2 h at 37°C. The plate was then washed with 0,01 M PBS containing 0,05% (v/v) Tween 20 (PBST). 100 µl of goat polyclonal anti-ApoA-I antibody (Acris, San Diego CA USA) diluted 1:6000 was mixed with serial dilution (1:1, 1:10, and 1:50) of rice seeds protein extraction (100 μl). From each mixture, 100 μl solution was added to each well coated, and the plate was incubated at 37°C for 1 h. The plate was washed with PBST and then a 1:5000 diluted solution (100 μl) of anti-goat IgG-HRP antibody (Thermofisher Scientific, Waltham Massachusetts USA) was added to each well and incubated at 37°C for 1 h. After that, the plate was rewashed with PBST, and a 50 μl of TMB was added to each well followed by incubation at 37°C for 15 min. To stop the reaction, 150 μl of 0,4 N hydrochloric acid (HCl) was added to each well, and then absorbance was measured at 450 nm using an ELISA plate reader (BIORAD, Hercules California USA). Each experiment was performed in triplicates. The standard curve using recombinant hAPOA-I (Sigma Aldrich, St. Louis Missouri USA) protein was constructed. (Supplementary Figure S3)
Immunofluorescence and Immunogold analyses
Rice seeds (mid-maturation) were collected, reduced in small pieces, and fixed in 50mM Hepes, pH 7,4, 2% formaldehyde, and 0,2% glutaraldehyde, overnight at 4°C. Samples were repeatedly rinsed in 50mM Hepes, pH 7.4, dehydrated with increasing concentrations of ethanol, and embedded in LR Gold resin (Sigma Aldrich St. Louis Missouri USA) at -20°C. Semi-fine sections (2µm) and ultra-thin sections (80 nm) were obtained using a Reichert Jung Ultracut E microtome. Ultra-thin sections were collected with nickel grids (Agar Scientific, Stansted United Kingdom) for immunogold experiments.
Semi-fine sections were hydrated with TBS (Tris-buffered saline, 20 mM TRIS, 150 mM NaCl pH 7.6) for 5 minutes, blocked with 1% BSA in TBS for 1 hour at room temperature, and then incubated with primary goat polyclonal anti-ApoA-I antibody (Acris, San Diego California USA) diluted 1:500 overnight at 4 °C. After three rinses in TBS, sections were incubated in the FITC-conjugated antigoat secondary antibody (Santa Cruz Biotechnology, Dallas Texas USA) at 1:200 in TBS for 2 hours at room temperature, rinsed three times with TBS, and mounted in Cityfluor. Negative controls were performed with only the secondary antibodies. Fluorescence observations were performed with a Leica DMRB microscope (set filter BP450-490, RKP 410, long pass 515). Images were acquired by a Leica MC170 HD camera.
Ultra-thin sections were hydrated with TBS for few minutes, blocked in BSA 1% in TBS for 30 min at room temperature, and incubated with primary goat polyclonal anti-ApoA-I antibody (Acris, San Diego California USA) at different dilution (from 1:500 to 1:200) in TBS at room temperature for 2 h. After three rinses in TBS, sections were incubated with 20 nm gold-conjugated rabbit anti-goat secondary antibody (purchased by BBA International, USA) diluted 1:100 for 1h at room temperature, rinsed three times with TBS, and post-fixed with 1% glutaraldehyde 15 min at room temperature. Sections were that stained with Uranyl acetate 3% for 20 min in the dark, rinsed with distilled water, dried, and observed with an EFTEM LEO 912AB transmission electron microscope (Zeiss, Oberkochen Germany) working at 100 kV.